RESUMEN
Plastidial acyl-acyl carrier protein:sn-glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the acyl-acyl carrier protein-dependent sn-1 acylation of sn-glycerol 3-phosphate (G3P) to produce lysophosphatic acid. Functional recombinant Erysimum asperum GPAT (EaGPAT), devoid of transit peptide, was produced in yeast. Analysis of the dependence of EaGPAT activity on increasing G3P concentration resulted in a hyperbolic response. EaGPAT exhibited a preference for 18-carbon unsaturated acyl-CoAs. Assays with concentrations of oleoyl-CoA up to 90µM revealed an exponential response to increasing concentrations of acyl donor, and the introduction of increasing concentrations of unlabeled linoleoyl-CoA into the standard reaction mixture resulted in increased incorporation of radiolabeled oleoyl moieties into lysophosphatidic acid. Collectively, the kinetic results suggest that acyl-CoA may act as both substrate and allosteric effector. EaGPAT was also shown to oligomerize to form higher molecular mass multimers, with the monomer and trimer being the predominant forms of the enzyme. Since most allosteric enzyme exhibit quaternary structure, the self-associating properties of EaGPAT are consistent with those of an allosteric enzyme. These results could have important regulatory implications when plastidial GPAT is introduced into a cytoplasmic environment where acyl-CoA is the acyl donor supporting cytoplasmic glycerolipid assembly.
Asunto(s)
Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/metabolismo , Glicerol-3-Fosfato O-Aciltransferasa/química , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Acilación , Regulación Alostérica , Secuencia de Bases , Proteínas de Cloroplastos/genética , Clonación Molecular , ADN de Plantas/genética , Erysimum/enzimología , Erysimum/genética , Glicerol-3-Fosfato O-Aciltransferasa/genética , Glicerofosfatos/metabolismo , Cinética , Filogenia , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Erysimum is a genus of the Brassicaceae family closely related to the genus Arabidopsis. Several Erysimum species accumulate 5ß-cardenolides. Progesterone 5ß-reductases (P5ßRs) first described in Digitalis species are thought to be involved in 5ß-cardenolide biosynthesis. P5ßRs belong to the dehydrogenase/reductase super-family of proteins. A full length cDNA clone encoding a P5ßR was isolated from Erysimum crepidifolium leaves by 5'/3' RACE-PCR (termed EcP5ßR). Subsequently, the P5ßR cDNAs of another nine Erysimum species were amplified by RT-PCR using 5' and 3' end primers deduced from the EcP5ßR cDNA. The EcP5ßR cDNA is 1170bp long and encodes for 389 amino acids. The EcP5ßR cDNA was ligated into the vector pQE 30 UA and the recombinant His-tagged protein (termed rEcP5ßR) was over-expressed in Escherichia coli and purified by Ni-chelate affinity chromatography. Kinetic constants were determined for progesterone, 2-cyclohexen-1-one, isophorone, and NADPH. The by far highest specificity constant (k(cat)K(M)⻹) was estimated for 2-cyclohexen-1-one indicating that this monocyclic enone may be more related to the natural substrate of the enzyme than progesterone. The atomic structure of rEcP5ßR was modelled using the crystal structure of P5ßR from Digitalis lanata 2V6G as the template. All sequence motifs specific for SDRs as well as the NFYYxxED motif typical for P5ßR-like enzymes were present and the protein sequence fitted into the template smoothly.